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IntroductionSystemic sclerosis (SSc) is a rare chronic autoimmune disease characterized by diffuse fibrosis of the skin and internal organs and vascular abnormalities. The etiology and physiopathology are complex due to the heterogeneity of its overall clinical presentation. Arsenic trioxide (ATO) has been proven to be effective against SSc, sclerodermatous Graft-versus-Host Disease, multiple sclerosis, Crohn’s disease or systemic lupus erythematosus animal models and has demonstrated promising effects in human clinical trials. Its efficacy was shown to be related at least in part to the generation of Reactive Oxygen Species (ROS) and the selective deletion of activated immune cells and fibroblasts. However, ATO can induce some adverse effects that must be considered, especially when used for the treatment of a chronic disease.MethodsWe evaluate here, in vitro and in a mouse model of SSc, the improved efficacy of ATO when associated with a Fenton-like divalent cation, namely copper chloride (CuCl2), also known to trigger the production of ROS.ResultsIn preliminary experiments in vitro, ATO 1 µM + CuCl2 0.5 µM increased ROS production and increased apoptosis of NIH 3T3 murine fibroblasts compared to 1 µM ATO alone. In vivo, in the HOCl-induced mouse model of SSc, co-treatment with ATO 2.5 μg/g + CuCl2 0.5 μg/g significantly alleviated clinical signs such as the thickening of the skin (p

Graft-versus Host Disease (GvHD) is a major complication of hematopoietic stem cell transplant. GvHD is characterized by the chronic activation of immune cells leading to the development of systemic inflammation, autoimmunity, fibrosis and eventually death. Arsenic trioxide (ATO) is a therapeutic agent under clinical trial for the treatment of patients with systemic lupus erythematosus (SLE) and chronic GvHD (cGvHD). This therapy is admittedly rather safe although adverse effects can occur and may necessitate short interruptions of the treatment. The aim of this study was to combine ATO with a divalent cation, to generate a Fenton or Fenton-like reaction in order to potentiate the deletion of activated immune cells through the reactive oxygen species (ROS)-mediated effects of ATO in a mouse model, and thereby enabling the use of lower and safer ATO concentrations to treat patients with cGvHD. In vitro, among the various combinations of divalent cations tested, we observed that the combination of ATO and CuCl2 (copper chloride) induced a high level of oxidative stress in HL-60 and A20 cells. In addition, this co-treatment also decreased the proliferation of CD4+ T lymphocytes during a mixed lymphocyte reaction (MLR). In vivo, in a cGvHD mouse model, daily injections of ATO 2.5 µg/g + CuCl2 0.5 µg/g induce a decrease in lymphocyte activation and fibrosis that was equivalent to that induced by ATO 5 µg/g. Our results show that the addition of CuCl2 improved the effects of ATO and significantly limited the development of the disease. This co-treatment could be a real benefit in human patients to substantially decrease the known ATO side effects and optimize ATO treatment in pathologies characterized by activated cells sensitive to an increase in oxidative stress.

Despite significant therapeutic advances, graft-versus-host disease (GvHD) remains the main life-threatening complication following allogeneic hematopoietic stem cell transplantation. The pathogenesis of GvHD is dominated by a dysregulated allogeneic immune response that drives fibrosis and autoimmunity in chronic forms. A multitude of cell therapy approaches, including infusion of myeloid cells, has been proposed to prevent GvHD through tolerance induction but yielded variable results. Myeloid cells like macrophages can be reprogrammed to develop adaptive-like features following antigenic challenge to reinforce or inhibit a subsequent immune response; a phenomenon termed ‘trained immunity’. Here we report that, whereas LPSlow-trained macrophages elicit a suppressor effect on allogeneic T cell proliferation and function in vitro in an IL-10-dependent manner, Bacille Calmette et Guérin (BCG)-trained macrophages exert an opposite effect. In a murine model of sclerodermatous chronic GvHD, LPSlow-trained macrophages attenuate clinical signs of GvHD with significant effects on T cell phenotype and function, autoantibodies production, and tissue fibrosis. Furthermore, infusion of LPSlow-macrophages significantly improves survival in mice with acute GvHD. Importantly, we also provide evidence that LPSlow-macrophages do not accelerate A20-lymphoma tumor growth, which is significantly reduced upon transfer of BCG-macrophages. Collectively, these data indicate that macrophages can be trained to significantly inhibit in vitro and in vivo allo-reactive T cell proliferation without exhibiting pro-tumoral effect, thereby opening the way to promising clinical applications.